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The cryopreservation of Xenopus sperm allows for a significant reduction of the number of animals that must be kept, more efficient archiving of genetically altered (GA) lines, and easy exchange of lines with other laboratories, leading to improvements in animal welfare and cost efficiency. In this protocol, sperm from Xenopus laevis or Xenopus tropicalis are frozen using straightforward techniques and standard laboratory equipment. Testes are macerated in Leibovitz's L-15 medium, mixed with a simple cryoprotectant made from egg yolk and sucrose, and frozen slowly overnight in a polystyrene box at −80°C. Unlike mouse sperm, Xenopus sperm can be stored at −80°C rather than in liquid nitrogen, further reducing costs. The frozen sperm are then used for in vitro fertilization.

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